Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen,Irrespective of APC Phenotype or Activation

Naïve anti-viral CD8⁺ T cells (T_CD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding T_CD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve T_CD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific T_CD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.     

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen

CD8⁺ T cells (T_CD8⁺) play a crucial role in immunity to viruses. Antiviral T_CD8⁺ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T_CD8⁺ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T_CD8⁺ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T_CD8⁺ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T_CD8⁺ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T_CD8⁺ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T_CD8⁺ response, allowing persistence of the peripheral virus.The induction of effector CD8⁺ T cells (T_CD8⁺) is a vital step in the eradication or control of many viral infections. The induction of antiviral T_CD8⁺ requires the presentation of virally derived peptides in complex with major histocompatibility complex (MHC) class I on the surface of specialized professional antigen-presenting cells (pAPC), most commonly a subset of dendritic cells (DC) that bear the CD8α chain (1, 29). The CD8α⁺ DC reside only in secondary lymphoid organs and not in the tissues, implying that cell-mediated transport or drainage of virus particles to a lymph node is required for initiation of a T_CD8⁺ response. Partial inhibition of DC migration from the skin can impair the initiation of a T_CD8⁺ response (2). After influenza infection in the lungs, there is a burst of DC migration, followed by a refractory period in which no DC migration occurs (19). The functional consequences of this refractory period of DC migration have not been explored.A number of viruses, particularly human papillomaviruses, infect the skin and are ignored by the immune response for extended periods of time (31). We sought to explore the possibility that, after a low-level peripheral virus infection of the skin, changes in DC migration may limit the availability of antigen in the draining lymph node and thus the induction of a T_CD8⁺ response. There are a number of confounding factors that make the study of DC migration in the initiation of an antiviral T_CD8⁺ response difficult. Virus particles may directly drain to the lymph node within seconds (11, 13, 25). In addition, many viruses will alter DC functions, including migration, after infection of the DC itself. This may occur via specific viral modulation of DC function (16) or via nonspecific shut down of host protein synthesis (26), both of which will affect migration. Thus, it is often not possible to distinguish between the effects of virus infection upon DC migration, drainage of virus directly to the lymph node, and the natural response that follows migration of DC responding to a peripheral virus infection.There is currently no mouse model of a peripheral virus infection that is confined to the skin, as no natural mouse papillomavirus has ever been isolated. Therefore, to address these issues, we have made use of another small DNA virus, namely, an adenovirus vector that is replication deficient (rAd). These vectors express influenza virus nucleoprotein (NP) under the control of a ubiquitous (cytomegalovirus [CMV] immediate-early) or tissue-targeted promoter (K14, targeted to keratinocytes, the site of papillomavirus replication). Antigen driven by the K14 promoter is expressed only in skin cells, so only uninfected DC can present antigen in this system, removing the need to account for modulation of the function of virus-infected DC.We demonstrate that when antigen is expressed in only keratinocytes in the skin, the efficiency of T_CD8⁺ induction is reduced and the time period for which antigen is available to prime effector cells is reduced dramatically. DC-mediated transport is required for antigen to reach the lymph node where a T_CD8⁺ response is initiated. The reduced time period of antigen presentation is the result of a transient blockade in DC migration from the site of infection. The blockade in DC migration reduced the delivery of viral antigen to the lymph node needed to induce a T_CD8⁺ response. The resulting T_CD8⁺ response to peripheral viral antigen is not capable of clearing all target cells presenting a viral peptide, thus allowing the persistence of peripheral virus-infected cells. These results provide a potential mechanism for the long-term evasion of the immune response by papillomaviruses following natural infection and also have important implications for tissue targeted gene therapy vectors.     

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.     

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

The gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8⁺ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8⁺ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8⁺ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8⁺ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle.     

Coxsackievirus B3 Inhibits Antigen Presentation In Vivo,Exerting a Profound and Selective Effect on the MHC Class I Pathway

Many viruses encode proteins whose major function is to evade or disable the host T cell response. Nevertheless, most viruses are readily detected by host T cells, and induce relatively strong T cell responses. Herein, we employ transgenic CD4⁺ and CD8⁺ T cells as sensors to evaluate in vitro and in vivo antigen presentation by coxsackievirus B3 (CVB3), and we show that this virus almost completely inhibits antigen presentation via the MHC class I pathway, thereby evading CD8⁺ T cell immunity. In contrast, the presentation of CVB3-encoded MHC class II epitopes is relatively unencumbered, and CVB3 induces in vivo CD4⁺ T cell responses that are, by several criteria, phenotypically normal. The cells display an effector phenotype and mature into multi-functional CVB3-specific memory CD4⁺ T cells that expand dramatically following challenge infection and rapidly differentiate into secondary effector cells capable of secreting multiple cytokines. Our findings have implications for the efficiency of antigen cross-presentation during coxsackievirus infection.     

Direct Presentation Is Sufficient for an Efficient Anti-Viral CD8+ T Cell Response

The extent to which direct- and cross-presentation (DP and CP) contribute to the priming of CD8⁺ T cell (T_CD8+) responses to viruses is unclear mainly because of the difficulty in separating the two processes. Hence, while CP in the absence of DP has been clearly demonstrated, induction of an anti-viral T_CD8+ response that excludes CP has never been purposely shown. Using vaccinia virus (VACV), which has been used as the vaccine to rid the world of smallpox and is proposed as a vector for many other vaccines, we show that DP is the main mechanism for the priming of an anti-viral T_CD8+ response. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines.     

T Cell Detection of a B-Cell Tropic Virus Infection: Newly-Synthesised versus Mature Viral Proteins as Antigen Sources for CD4 and CD8 Epitope Display

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8⁺ and CD4⁺ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8⁺ and CD4⁺ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36–48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4⁺ T cells.     

Cross-presentation of epitopes on virus-like particles via the MHC I receptor recycling pathway

Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.     

Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

Three-dimensional structure determines the pattern of CD4+ T-cell epitope dominance in influenza virus hemagglutinin

The structural context of a CD4⁺ T-cell epitope is known to influence immunodominance at the level of antigen processing, but general rules have not emerged. Dominant epitopes of influenza virus hemagglutinin are found to be localized to the C-terminal flanks of conformationally stable segments identified by low crystallographic B-factors or high COREX residue stabilities. The bias toward C-terminal flanks is distinctive for antigens from the influenza virus. Dominant epitopes in antigens/allergens from other sources also localize to the flanks of stable segments but are found on either N- or C-terminal flanks. Thus, dominance arises from preferential endoproteolytic nicking between stable segments followed by loading of fragment terminal regions into antigen-presenting proteins. This mechanism probably arose in order to direct CD4⁺ responses onto sequences that are conserved for structure and function. Structure-guided presentation could enhance protection against genetically drifting influenza virus variants but most likely reduces protection against new viral subtypes.     

Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+ Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design

The role of CD4⁺ and CD8⁺ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4⁺- and CD8⁺-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8⁺- or CD4⁺-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4⁺-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4⁺ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.There is now increasing evidence to suggest that both CD4⁺ and CD8⁺ T cells are critical for the generation of an effective immune response against intracellular pathogens. Although both CD4⁺ and CD8⁺ T cells recognize nonnative forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of antigen to these two types of T lymphocytes occurs through distinct pathways (24). In fact, the disparity in antigen presentation to these T cells is not due to processing differences but rather reflects the differences in the capacities of class I and class II molecules to bind antigenic determinants in an intracellular compartment. Indeed, earlier studies have shown that for processing and interaction with MHC class II molecules, antigen expressed de novo needs to be targeted to an endosomal or lysosomal compartment (5). There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments (3, 8, 26, 41). On the other hand, class II-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens which are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface (11). Although, in certain experimental systems, cytoplasmic and nuclear proteins may also enter this endogenous pathway, generally these proteins are targets for the class I processing pathway (9, 14, 20, 27).One of the major limitations of the epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs (2, 35, 38). Based on these observations, we reasoned that a molecular approach that directly routes these epitopes into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4⁺ T cells. The lysosome-associated membrane protein (LAMP-1) and the invariant chain (Ii) are transmembrane proteins which are localized predominantly in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to the specific compartments. We therefore designed a chimeric polyepitope construct capable of encoding multiple class II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1 and the Ii protein, with the aim of targeting the epitopes to the endosomal and lysosomal compartments. The data presented in this study clearly demonstrate that if the endogenously synthesized polyepitope protein is targeted to the endocytic/secretory pathway, processing and presentation of all the epitopes are dramatically enhanced. More importantly, minimal epitope sequences, without any natural flanking sequences, were adequate for efficient stimulation of the virus-specific memory CTL response, a result that has important implications for epitope-based vaccine design.     

Human macrophages and dendritic cells can equally present MART-1 antigen to CD8(+) T cells after phagocytosis of gamma-irradiated melanoma cells

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8⁺ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8⁺ T cell clone. Confocal microscopy with Alexa Fluor®⁶⁴⁷-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8⁺ T cell cross-presentation thereafter.     

Manipulating IL-2 Availability Amid Presentation of Donor MHC Antigens Suppresses Murine Alloimmune Responses by Inducing Regulatory T Cells

Background

Major histocompatibility complex (MHC) antigens are important for alloimmune responses as well as immune tolerance. Previous studies have shown that presentation of donor MHC antigens by donor-specific transfusion prior to or upon transplantation promotes transplant tolerance induced by other agents. However, it is unclear whether presentation of donor MHC antigens by DNA vaccination induces long-term allograft survival.

Methodology/Principal Findings

We investigated whether presentation of MHC class-II and/or class-I donor antigens by DNA vaccination suppresses alloimmune responses and promotes long-term allograft acceptance. We initially found that presentation of both MHC donor antigens by DNA vaccination itself prior to transplantation fails to significantly prolong islet allograft survival in otherwise untreated mice. However, islet allograft survival was significantly prolonged when MHC class-II DNA vaccination was accompanied with IL-2 administration (MHCII + IL-2) while MHC class-I DNA vaccination was followed by IL-2 and subsequent neutralizing anti-IL-2 treatments (MHCI + IL-2/anti-IL-2). Especially, this protocol promoted long-term allograft survival in the majority of recipients (57%) when combined with low doses of rapamycin post-transplantation. Importantly, MHCII + IL-2 induced FoxP3+ Treg cells in both spleens and grafts and suppressed graft-infiltrating CD4+ cell proliferation, whereas MHCI + IL-2/anti-IL-2 mainly inhibited graft-infiltrating CD8+ cell proliferation and donor-specific CTL activity. The combined protocol plus rapamycin treatment further reduced both CD4+ and CD8+ T cell proliferation as well as donor-specific CTL activity but spared FoxP3+ Treg cells. Depleting CD25+ Treg cells or adoptive transfer of pre-sensitized CD8+ T cells abolished this long-term allograft survival.

Conclusions/Significance

Manipulating IL-2 availability during presentation of MHC class-II and class-I donor antigens by DNA vaccination pre-transplantation induces Treg cells, suppresses alloimmune responses and promotes long-term allograft survival.     

Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (T_regs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. T_reg abnormalities have been associated with several autoimmune diseases and there is interest in the role of T_regs in SLE. We previously demonstrated that transfer of expanded CD4⁺CD25⁺CD62L^HI T_regs slows the development of lupus in (NZBxNZW)F₁ (B/W) mice. However in the absence of T_reg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3⁻ effector T cells (T_effs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure T_reg subset defined by CD4⁺CD25⁺ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4⁺CD25⁺ cells produced a population containing 70–85% CD4⁺Foxp3⁺T_regs. Expanded T_regs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4⁺CD25⁺ T_regs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of T_eff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred T_regs or transferred T_effs expanded in the absence of T_regs. These studies demonstrate that adoptive transfer of expanded CD4⁺CD25⁺Foxp3⁺T_regs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured T_eff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated T_effs from autoimmune patients may not pose a significant risk of promoting disease.     

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.     

Preserved central memory and activated effector memory CD4+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP36 study

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV type 1 replication for 10 years or more in the absence of antiretroviral treatment. In the present study, HIV controllers (n = 11) maintained potent HIV-specific CD4 responses in spite of very low antigenic loads. Their CD4⁺ central memory T (T_CM) cells were characterized by near-normal numbers and preserved interleukin-2 (IL-2) secretion in response to HIV antigens and uniformly high expression of the survival receptor IL-7 receptor α (IL-7Rα). Controllers expressed CCR7 at higher levels than uninfected controls, suggesting differences in T_CM-cell homing patterns. CD4⁺ effector memory T (T_EM)-cell responses were polyfunctional in HIV controllers, while IL-2 secretion was lost in viremic patients. Cytokine production was three times higher in controllers than in treated patients with undetectable viral loads, suggesting an intrinsically more efficient response in the former group. The total CD4⁺ T_EM-cell pool underwent immune activation in controllers, as indicated by increased HLA-DR expression, decreased IL-7Rα expression, a bias towards gamma interferon production upon polyclonal stimulation, and increased macrophage inflammatory protein 1β secretion associated with chronic CCR5 down-regulation. Thus, HIV controllers showed a preserved CD4⁺ T_CM-cell compartment and signs of potent functional activation in the CD4⁺ T_EM-cell compartment. While controllers did not show the generalized immune activation pattern associated with disease progression, they had signs of immune activation restricted to the effector compartment. These findings suggest the induction of an efficient, nondetrimental type of immune activation in patients who spontaneously control HIV.     

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.